The present invention relates in general to methods and compositions for stabilizing coenzymes and in particular to methods and compositions for stabilizing NADH in solution.
Biochemical reactions are almost universally catalyzed by enzymes. Each enzyme is a protein which promotes a highly specific chemical change in a substrate.
In order for many kinds of enzymes to function, the participation of a type of low molecular weight molecule, called a coenzyme, is required. In general, a chemical change in a coenzyme counterbalances a change in a substrate which is the desired outcome of a reaction. For example, a coenzyme may accept a hydrogen ion from or donate a hydrogen ion to a substrate.
A number clinical of diagnostic assays involve oxidation-reduction reactions. Among these diagnostic reactions are those in which a dinucleotide acts as a coenzyme. Examples of such dinucleotides include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide 2'-phosphate (NADP) and flavin adenine dinucleotide (FAD).
Dinucleotides are mononucleotides joined by a phosphate bridge. A mononucleotide is a phosphoric acid ester of nitrogenous base and a sugar. In NAD and NADP, a first mononucleotide is a phosphoric acid ester of a nucleoside formed from the base adenine and the sugar ribose, while a second mononucleotide is a phosphoric acid ester of a nucleoside formed from the base nicotinamide and ribose. In FAD, a first mononucleotide formed from a phosphoric acid ester of adenine and ribose is linked to a second mononucleotide formed from a phosphoric acid ester of the base 7,8-dimethylisoalloxazine and the sugar alcohol D-ribitol.
Either NAD or NADP may serve as an electron acceptor from a reduced substrate (S.sub.r) by receiving a hydrogen with associated electrons or may serve as an electron donor to an oxidized substrate (S.sub.o) in a reverse reaction, i.e.: EQU NAD.sup.+ +S.sub.r -H.sub.2 =NADH+S.sub.o +H.sup.+ ( 1) EQU NADP.sup.+ +S.sub.r -H.sub.2 =NADPH+S.sub.o +H.sup.+ ( 2)
A useful feature of these reactions is that the reduced forms of these dinucleotides (i.e., NADH and NADPH) absorb light at a wavelength of 340 nm, but the oxidized forms (i.e., NAD.sub.+ and NADP.sub.+) do not. Therefore, after plotting a calibration curve of reaction rate for a known quantity of enzyme, the unknown quantity of an enzyme catalyzing one of these reactions may be obtained from a given amount of substrate, a known activity for the enzyme and an observed rate of change in optical density at 340 nm. Likewise, the quantity of a substrate of an enzyme catalyzing one of these reactions may be determined from the calibration curve, a given amount of an enzyme of known activity and a measured rate of change in optical density at 340 nm.
Although valuable in diagnostic assays, NADH, like other reduced dinucleotides, is very unstable in aqueous solution. This presents a particular problem for the manufacturer of and for users of diagnostic assay kits inasmuch as it is much easier, cheaper and more accurate to dispense an aqueous solution of NADH than it is to dispense NADH in the more stable form of a dry powder.
One approach to stabilizing an NADH solution involves mixture with an organic solvent to eliminate as much water as possible. In U.S. Pat. No. 4,153,511, an inert, hygroscopic agent and an organic solvent are employed to obtain an NADH solution containing less than 0.5% water. However, NADH solutions in available non-aqueous solvents tend to be viscous to the point of being difficult to dispense accurately and precisely.
In another approach, coenzymes, such as NAD and NADP, are stabilized in the presence of an organic solvent, preferably a liquid polyol such as glycerol or propylene glycol, at an acidic pH. However, such solutions are relatively unstable and are particularly unsuitable for automated assays where virtually 100% stability of the reduced coenzyme is required.